Download Mitochondrial Viability ab129732

ab129732 –
Mitochondrial Viability
Assay
Instructions for Use
For measuring mitochondrial/cellular viability in
high throughput
This product is for research use only and is not
intended for diagnostic use.
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Table of Contents
1.
Introduction………………………………………………………….3
2.
Assay Summary…………………………………………………….5
3.
Kit Contents………………………………………………………….6
4.
Storage and Handling………………………………………………6
5.
Additional Materials Required……………………………………..6
6.
Preparation of Reagents…………………………………………..7
7.
Sample Preparation………………………………………………..7
8.
Assay Procedure…………………………………………………..8
9.
Data Analysis………………………………………………………9
10. Assay Performance and specificity………………………………9
11. Frequently Asked Questions…………………………………….14
12. Troubleshooting……………………………………………………17
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1. Introduction
Principle: ab129732 contains a fluorometric/colorimetric dye used to
determine the mitochondrial viability of live cells in a high throughput
format. It uses an indicator dye (7-Hydroxy-3H-phenoxazin-3-one 10oxide) to measure oxidation-reduction reactions which principally
occur in the mitochondria of live cells. When reduced by
metabolically active cells the non-fluorescent dark blue dye becomes
fluorescent pink with absorbance at 570nm and red-fluorescent
properties (560±10Ex/590Em) at neutral pH. The dye can be
measured
in
fluorescence
or
absorbance
mode.
However
fluorescence mode measurement offers greater assay linearity,
reproducibility, robustness and sensitivity.
Background:
Oxido-reductase reactions are carried out mostly by mitochondrial
enzymes in the cell. Mitochondrial enzymes reduce the viability stain
in this kit to a pink fluorescent counterpart. This assay can be used
to discriminate between compounds which affect overall cellular
metabolism
mitochondria.
and
viability
from
those
that
specifically
affect
This can be achieved by growing cells in media
supplemented with two different carbon sources. The mechanism
and details of this approach are given below.
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When cells are cultured in vitro using glucose rich media, many
compounds known to be toxic to mitochondria and in particular the
electron transport chain (ETC) will affect minimally cellular viability
even at very high concentrations.
This is because the cells will
continue to generate ATP through glycolysis, preventing cell death
from occurring. However, when cells are cultured in galactose and
glutamine, the cellular metabolism remains dependent upon
mitochondrial function and therefore toxic compounds to the ETC will
rapidly deplete ATP leading to cell death.
There is much controversy in the mitochondria field as to the
mechanism of this dependence.
Likely the inter-conversion of
galactose to glucose is a high energy consuming pathway.
The
consumption of energy to generate one molecule of glucose from
galactose is as costly as the amount of energy generated by that
molecule of glucose via the glycolysis pathway.
Therefore,
glycolysis alone will yield a net of zero ATP. Instead the major
source of energy is the metabolism of glutamine supplemented in the
media. The glutamine is oxidized via the citric acid cycle to generate
NADH
for
the
respiratory
chain.
Prolonged
exposure
to
galactose/glutamine induces up-regulation of OXPHOS components
indicating the increased dependence upon mitochondrial function
during these culture conditions.
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2. Assay Summary
Seed cells in a microwell culture plate.
Carry out experimental conditions under which cellular/mitochondrial
viability is to be assessed
Once the time course of experimental conditions has been
completed, add stain to the culture media
Incubate for 4 hours at 37˚C.
Read by fluorescence of absorbance
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3. Kit Contents
Reagent is provided for more than 2000 assays on 96-well plates.
20x Mitochondrial viability stain
24 mL
4. Storage and Handling
Store Mitochondrial viability stain upright protected from the light at
4°C.
For frequent use, this product may be store at room
temperature for 1 week. This kit is stable for at least 6 months from
receipt.
5. Additional Materials Required

A standard spectrophotometer capable of 570 nm endpoint
reading or a fluorescent reader capable of 550 nm excitation
and 590 nm emmision

Dark wall 96 or 384-well plate(s).

Cell culture reagents.

Multi and single channel pipettes.
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6. Preparation of Reagents
6.1
Equilibrate stain to room temperature.
6.2
Optional: Prepare a 2X Mitochondrial viability stain
solution by diluting the 20x stock 10 fold (i.e. for 1 96-well
plate dilute 1mL of 20X Mitochondrial viability stain in 9mL
of growth media). Mix well. Store in the dark at room
temperature. Reagent is provided for at least 2000 tests,
each carried out in a total volume of 200 µL per well.
Note = Adding 100 µL per well of a 2X solution will
increase reproducibility of the assay over addition of 5 µL
of a 20X stock solution.
7. Sample Preparation
Note: The protocol below is described for a 96-well plate. If
performing the assay on a 384-well plate, adjust volumes
accordingly.
This assay has been optimized for use on
adherent and suspension cells.
7.1
Seed cells directly into a dark walled 96-well plate.
If
seeding adherent cells allow attachment for several hours to
overnight. It is advisable to seed in a 100 µL volume of the
same media used to maintain the cells in bulk culture. The
optimal cell seeding density is dependent on cell type and
duration of experimental time course.
Experimental
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examples and cell seeding are shown in the data analysis
section below. Details about galactose/glutamine cultured
conditions are shown in FAQs section.
7.2
Ensure that there will be at least one well per experimental
condition to which cells but no dye and also one well per
plate with dye, to assess background signal.
8. Assay Procedure
8.1
At the end of the treatment or experimental condition time
course, overlay 100 µL of 2X Mitochondrial viability stain on
each well containing 100 µL of media and incubate under
sterile conditions at 37˚C for 4 hours.
Note = 4 hour incubation is long enough to give a robust and
reproducible signal, yet is short enough to prevent added
toxic cellular effects due to the dye itself.
8.2
Read the plate by absorbance at 570nm or by fluorescence
and set the instrument with 550nm excitation and 590nm
emission.
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9. Data Analysis
Subtract media/experimental condition background (test wells
without dye) from the actual test measurements prior to subtracting
background (empty wells with dye) from all the measurements.
10.
Assay Performance and specificity
Effect of Mitochondrial viability stain incubation time on
fluorescent signal formation – The Mitochondrial viability stain has
been typically used after incubation from 2 to 48 hours. Optimal
reproducibility with the lowest background is obtained after 4 hours
of incubation in most cell lines. Caution must be exercised when
changing the incubation conditions specified in this protocol, since
longer incubation times have shown to induce oxidative stress by the
dye itself in certain cell lines.
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Figure 1. Effect of stain incubation time on assay reproducibility and
background. Amine coated dark wall plates were seeded in a titration
series of each cell line in a final volume of 100 µL of media.
2X
mitochondrial viability stain (diluted in the cell line specific growth media)
was added to each well at specified time points. Cartesian (XY) graph data
is shown after media and background subtraction. Bar graph shows mean
background signal at different time points. 4 hour incubation of stain results
in optimal reproducibility with minimal increase of background over the 2
hour incubation. Reproducibility was calculated for Jurkat cells, HepG2 cells
and HeLa cells. %CV after 4 hours of incubation was 10%, 7% and 2.4%
respectively, whereas %CV after 2 hours of incubation was 11%, 33% and
6.1% respectively.
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Suitability of the mitochondrial viability assay for highthroughput screening –Fluorescent and colorimetric readout Z
factors were assessed in Jurkat and HepG2 cells, respectively.
Average Z factors for both readouts was greater than 0.7.
Figure 2. Dynamic range and suitability of the mitochondrial viability
assay for high throughput screening. Cells were seeded in a titration
series with n=11 for each data point. Fluorometric readout gave an average
Z factor of 0.82 on Jurkat cells seeded from 1,500 – 1000,000 cells per well
on a 96-well plate. Colorimetric readout gave an average Z factor of 0.72 on
HepG2 cells seeded from 40,000 – 150,000 cells per well.
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Mitochondrial viability stain as an indicator of cellular viability –
This assay was validated as an indicator of cellular viability after
dose-response treatment of Jurkat cells with idarubicin and
staurosporin. Idaribicin is an antileukemic drug which inserts itself
into DNA and interferes with topoisomerase II, inducing rapid cell
death. Staurosporin is a non-specific kinase inhibitor that is known
to induce apoptosis in many cell lines.
Figure 3. Jurkat cells were seeded at 25,000 cells per well in a 50 µL
volume and were immediately overlay with a 2X concentration of either
Idarubicin or Staurosporin for a final volume of 100 µL.
Cells were
incubated for 2 hours with Idarubicin and for 4 hours with Staurosporin prior
to the addition of 2X stain diluted in RPMI media. After 4 hours of further
incubation with stain, fluorescence was measured. IC50 for Idarubicin was
found at 22 – 25 µM and IC50 for staurosporin was found at 500nM.
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Mitochondrial viability stain as an indicator of the mitochondrial
metabolic state – This assay was further validated in the context of
mitochondrial toxicity, comparing fluorescent readout after 72 hour
rotenone treatment in HepG2 cells cultured in glucose versus
galactose/glutamine.
Figure 4. Mitochondrial viability stain in long term toxicity. HepG2
cells previously cultured in glucose or galactose/glutamine supplemented
media were seeded at 10,000 cells per well and allowed to adhere
overnight.
Media was replaced for the specific culture media in the
presence of a titration series of Rotenone (5 µM – 0.5 pM). Cells were
treated for 72 hours. Cells were incubated with 2X stain for 4 hours before
fluorescent readout analysis.
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11.
Frequently Asked Questions
11.1
I want to use a cell line other than the cell lines shown in
section 10 of this protocol, how do I know how many cells to
seed per well?
The cell seeding density is dependent on the cell type, the time
course in which experimental conditions will be evaluated and
instrument capabilities. As a rule of thumb, highly metabolic cells (i.e.
HepG2) require lower seeding than low metabolic cells (i.e. Jurkat).
Furthermore, fluorometric reading allows for greater flexibility in
seeding due to a wider dynamic range and increased sensitivity over
colorimetric reading (which can easily saturate at the high end of the
assay). To optimize for all these variables, we suggest seeding a
two-fold dilution series of cells (12 points) starting from 100 –
200k/well and down to zero. Incubate the cells for the same duration
as the proposed treatment then stain as described. Once this data is
analyzed, the experimental plate should be seeded near the top of
the linear range.
11.2
Can I incubate stain for a different amount of time?
Yes, staining is typically observed between 2 and 48 hours of
incubation.
However, we have found that 2 hours of incubation
leads to low reproducibility. Any metabolic cell staining procedure
will affect the cells therefore we do not recommend treatments of
longer than 6 hours. Long treatments alone may trigger cellular
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reactive oxygen species leading to mitochondrial dysfunction,
reduced proliferation and autophagy.
11.3
How do I treat cells with Galactose media?
Culture the cell line of interest in the following media: DMEM without
glucose containing 5mM D-Galactose, 6mM L-glutamine, 1mM
sodium pyruvate, 44mM sodium bicarbonate, 1X RPMI non-essential
amino acids, 10% Dialyzed FCS and 1X Penicillin/streptomycin. It is
advisable to generate a stock solution of 1M D-Galactose in media
without glucose prior to generating the complete galactose media.
The 1M D-Galactose stock solution may require warming at 37˚C for
complete solubility. The galactose stock can be stored long term at 20°C. Cells cultured in galactose may decrease in doubling time
compared
to
glucose
counterparts.
Ensure
that
the
galactose/glutamine cultured cells are split at a maximum of 1:2, to
maintain a healthy culture.
11.4
My cells do not adhere well to the standard tissue culture
treated 96-well plate?
To improve on adherence of cells, it may be necessary to seed cells
on plates with specialized coating surfaces, such as collagen, PolyL-Lysine, amine or carboxyl surfaces, however this assay can also
be performed on suspension cells
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11.5
I need to determine mitochondria health, should I use the
ATP assay or should I use the mitochondrial viability stain
assay?
The
mitochondrial
viability
stain
when
used
with
the
galactose/glutamine media is particularly useful for determining
mitochondrial health after a prolonged/chronic treatment (i.e >48
hours). If short incubation times/acute effects are to be measured
(i.e. less than 6 hours) we recommend measuring ATP assay
(ab113849).
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12.
Troubleshooting
Problem
High
background
Saturation of
signal by
fluorescence
reading
High CV
Low Signal
Cause
Product has
been exposed to
light for
extended
periods of time
Plate reader
lamp intensity is
too high
Staining time too
brief
Staining time too
brief
Inadequate
reagent volumes
or improper
dilution
Solution
Do not expose the dye
to direct light
Decrease the lamp
intensity or decrease
seeding of cells and
ensure to work within
the dynamic range of
the assay
Increase stain
incubation to 4 hours
Increase stain
incubation to 4 hours
Check pipettes and
ensure correct
preparation
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All information / detail is correct at time of going to print.
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